Sepsis afflicts -500,000 Americans per year with an associated mortality of 35-65%. Lipopolysaccharide (LPS), the predominant integral outer membrane component of all Gram-negative bacteria, is a principal initiating stimulus of an inflammatory cascade that has been referred to as the "Systemic Inflammatory Response Syndrome (SIRS)." This type of uncontrolled inflammatory response often leads to shock, and ultimately, death. During the past 4 years of funding on this grant, we identified the proteasome as a key regulator of LPS-induced signaling through Toll-like receptor 4 (TLR4) and have extensively analyzed the contribution of this important organelle to LPS-induced inflammatory responses in macrophages. The long-range goal of this research program is to define the molecular mechanisms of pathogenesis of septic shock and to identify potential therapeutic intervention strategies to reduce morbidity and mortality resulting from this spectrum of diseases. Our objective in this application is to elucidate mechanisms of regulation of LPS induced inflammation in macrophages by the proteasome. Our central hypothesis is that the proteasome serves as a key regulator of the inflammatory process and figures centrally in LPS-induced, Toll-like receptor (TLR) 4-mediated signaling. We further hypothesize that inhibition of the proteasomal proteases, either by specific inhibitors in vivo, or by selected mutations in genes that encode these proteases in mice in vivo, will modulate LPS-induced pro-inflammatory gene expression and lead to the identification of new targets for septic shock. The following specific aims have been proposed: Specific Aim 1: To establish the contribution of individual proteasomal proteases to LPS-induced inflammatory responses (in macrophages). Specific Aim 2: To identify mechanisms by which proteasomes contribute to regulation of LPS-induced signal transduction. Specific Aim 3: To develop a proteasome- and microbial- based therapeutic intervention to treat experimental septic shock. We anticipate that, at the completion of these Specific Aims we will 1) have determined mechanisms by which proteasomes contribute to LPS-induced signal transduction, 2) have a significantly better understanding of the role of the proteasome in LPS-induced signal transduction in macrophages, and 3) developed novel strategies for prevention of septic shock.